Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen that remains a serious health threat worldwide. Resistance of S. pneumoniae to multiple antibiotics is increasing at an alarming rate, and the population of immunocompromised individuals who are at risk for pneumococcal invasive diseases continues to grow. Relatively little is known about the roles played by pneumococcal physiology and metabolism in promoting colonization and virulence of its human host. The overall goal of this grant is to explore the roles played by inorganic phosphate (Pi) uptake and its regulation in the pathogenesis, antibiotic resistance, and metal ion homeostasis of S. pneumoniae. Pi uptake has already been implicated in pneumococcal invasive disease, but little is known about the mechanisms underlying this involvement. Several pieces of evidence show that the regulation of Pi transport in S. pneumoniae is fundamentally different from the mechanisms determined previously for model bacteria, like E. coli and B. subtilis. Therefore, this grant will challenge te current paradigms of Pi uptake that are based on these model bacterial systems and relate Pi uptake to the pathogenesis of S. pneumoniae for the first time. In addition, this work will validat and explore a new mechanism of pneumococcal -lactam resistance about which nothing is known. Finally, this grant draws together several recent ideas from the scientific literature to formulate the hypothesis that cellular Pi uptake and Pi cellular amount are tied to the availabilit and toxicity of several key divalent cations, including zinc (Zn2+) and manganese (Mn2+). Four Specific Aims will be met by this grant. Aim 1 will determine whether two predicted Pi ABC transporter pumps (designated as Pst1 and Pst2) separately uptake Pi in S. pneumoniae and whether this uptake is coordinated. Another goal of Aim 1 is to develop a powerful genetic toolbox and a conceptual framework about Pi uptake and its regulation that will allow effective exploration of the topics in Aims 2-4. Aim 2 will determine the roles of the Pst1 and Pst2 Pi transporters and the likely regulators (designated as PnpRS, PhoU1, and PhoU2) in pneumococcal colonization and virulence. Aim 2 will also determine whether there are additional routes of Pi uptake during infection, besides Pst1 and Pst2. Aim 3 will determine whether increased expression of the Pst1 transporter is responsible for resistance to -lactam antibiotics and whether the Pst2 transporter also plays a role. Aim 4 will explore whether there are links between Pi uptake and cellular amount and divalent metal ion homeostasis. There is a high likelihood of obtaining publishable data from this work that will serve as the basis for a future RO1 application and open up a significant new research area in S. pneumoniae. In addition, this grant has the potential to provide new targets for antibiotic and vaccine development and to provide information about an uncharacterized mechanism of -lactam antibiotic resistance in this important opportunistic pathogen.